Deoxyribonuclease IIβ proteins and CDNAs

ABSTRACT

The present invention provides cDNAs encoding deoxyribonuclease IIβ and isolated, purified deoxyribonuclease IIβ proteins. Antibodies against this protein and antisense agents targeted to a cDNA or corresponding mRNA encoding deoxyribonuclease IIβ are provided. In addition, methods of identifying and using modulators of deoxyribonuclease IIβ activity are described.

This case is a continuation-in-part to case Ser. No. 09/574,942, filedMay 19, 2000, now U.S. Pat. No. 6,358,723, which is a Continuation inpart of case Ser. No. 09/541,840, filed Apr. 3, 2000, now abandoned.

INTRODUCTION

This invention was made in the course of research sponsored by theNational Institutes of Health. The U.S. Government may have certainrights in this invention.

BACKGROUND OF THE INVENTION

Controlled cell death is critical for the life of a human; too much celldeath can cause the symptoms of cystic fibrosis and also lead todiseases such a neurodegeneration and acquired immune deficiencysyndrome (AIDS). In contrast, too little cell death can lead to canceror autoimmune diseases. Recent studies have defined the pathway of celldeath as “apoptosis” and have identified some of the biochemical stepsinvolved.

Apoptosis is a homeostatic mechanism involved in the controlled death ofobsolete cells during metamorphosis, differentiation, cell turnover, andhormone mediated deletion of thymocytes (Wyllie et al. Int. Rev. Cytol.1980 68:251-306). Apoptosis has also been identified as the mechanism ofcell killing during growth factor withdrawal (Rodriguez-Tarduchy et al.EMBO J. 1990 9:2997-3002; McConkey et al. J. Biol. Chem. 1990265:3009-3011), T-cell deletion, treatment with many cytotoxic agents(Cohen, J. J. and Duke, R. C. J. Immunol. 1984 132:38-42; Barry et al.Biochem. Pharmacol. 1990 40:2353-2362; Kaufmann, S. H. Cancer Res. 198949:5870-5878; and McConkey et al. Science 1988. 242:256-259), andfollowing hyperthermia (Barry et al. Biochem. Pharmacol. 199040:2353-2362; Lennon et al. Biochem. Soc. Trans. 1990 18:343-345; Takanoet al. J. Pathol. 1991 163:329-336).

Central to the mechanism of apoptosis is internucleosomal DNA digestionby endogenous endonucleases. Mammalian cells contain a variety ofendonucleases which could be involved in internucleosomal DNA digestion.It was originally postulated that the primary endonuclease involved inapoptosis is a Ca²⁺/Mg²⁺-dependent endonuclease. SeveralCa²⁺/Mg²⁺-dependent endonucleases have been identified, one of which isdeoxyribonuclease I (DNase I), (Peitsch et al. EMBO J. 1993 12:371).

Recent experiments, however, indicate that DNase I may not be theprimary endonuclease involved in apoptosis. It has been found that manycells do not contain this endonuclease. The role of DNase I, or anyother Ca²⁺/Mg²⁺-dependent endonuclease is further unlikely, as often noincrease or only a minor increase in Ca²⁺ levels occurs in apoptoticcells (Eastman, A. Cell Death and Differentiation 1994 1:7-9).

An alternate endonuclease that is active below pH 7.0 and has noapparent requirement for Ca²⁺ or Mg²⁺ has been detected (Sorenson etal., J. Natl Cancer Inst. 1990 82:749). This alternate endonuclease wasidentified as deoxyribonuclease II (DNase II; Barry, M. A. and Eastman,A. Archives of Biochem and Biophys. 1993 300(1):440-450). It wasproposed that this enzyme is involved in the internucleosomal digestionor fragmentation of DNA which is one of the early steps in the pathwayof apoptosis. Another report that has implicated DNase II in cell deathinvolves lens fiber cell differentiation, a process where the cells losetheir nuclei in a manner similar to apoptosis (Torriglia, A. et al. 1995J. Biol. Chem. 270:28579-28585). In this process, the chromatincondenses and the cells degrade their genomic DNA. DNase II was found byimmunocytochemistry to be localized in the cytoplasm but translocated tothe nucleus of the fiber cell before degeneration. These findingsimplicate DNase II as the endonuclease responsible for genomicdegradation observed during lens nuclear degeneration, and furthersupport a role for this enzyme in mechanisms of cell death.

However, more recent results have implicated yet another endonuclease,referred to CAD or caspase-activated deoxyribonuclease, in apoptosis(Enari, M. et al. 1998 Nature 391:43-50). Thus, it remains to bedetermined which specific endonuclease is involved in apoptosis,

The enzyme referred to herein as deoxyribonuclease IIα (DNA IIα) wasisolated and purified and the amino acid sequence determined(PCT/US97/18262). The DNA sequences for both the human and bovineproteins of DNase IIα have also been cloned (PCT/US97/18262). Use of DNAIIα in alleviating the suffering in patients with cystic fibrosis isalso disclosed in this PCT application.

In cystic fibrosis, the lungs of patients fill with the remnants of deadcells, and in particular with the DNA from these dead cells. Thepresence of DNA makes the mucous plugs too viscous to expel. A suggestedtherapy for these symptoms is the use of DNase I to digest the DNA,thereby permitting expulsion of the mucous plugs. However, this therapyhas not been particularly effective due to inactivity of the DNase Ienzyme in the presence of actin, also present in the sputum.

It is believed that DNase II enzymes and variations thereof may providea more effective therapeutic alternative.

Another isoform of the DNase II enzyme, referred to herein asdeoxyribonuclease IIβ (DNase IIβ) has now been identified and the geneand protein sequences for the mouse and human homolog have beendetermined.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a cDNA encodingdeoxyribonuclease IIβ.

Another object of the present invention is to provide an isolated,purified deoxyribonuclease IIβ enzyme.

Yet another object of the present invention is to provide antibodiesagainst this protein which can be used in diagnosing cells at variousstages in the apoptotic pathway.

Yet another object of the present invention is to provide antisenseagents targeted to a cDNA or corresponding mRNA encodingdeoxyribonuclease IIβ.

Yet another object of the present invention is to provide a method foridentifying agents that inhibit DNase IIβ activity comprising treatingcells with a test agent, transfecting cells with DNase IIβ, maintainingsaid transfected cells in culture, and monitoring apoptosis in treatedand untreated cells to determine whether the test agent modulatesapoptosis.

Yet another object of the present invention is to provide a method forinducing apoptosis in selected cells comprising transfecting cells witha vector expressing the DNase IIβ cDNA so that apoptosis is induced.

Yet another object of the present invention is to provide a method ofdigesting DNA released from dead cells with an effective amount of anisolated, purified DNase IIβ protein so that DNA is digested.

DETAILED DESCRIPTION OF THE INVENTION

The existence of a deoxyribonuclease II (DNase II) enzyme as a proteinof lysosomal origin that is involved in cellular digestion of foreignDNA has been known for many years. Recently, a DNase II enzyme has beenlinked with the DNA fragmentation that occurs at an early stage inapoptosis. The bovine and human forms of this DNase II protein, referredto herein as DNase IIα protein have been isolated and purified and theamino acid sequences of these proteins are disclosed in PCT/US97/18262.cDNAs encoding the bovine and human form of DNase IIα have also beencloned and characterized in PCT/US97/18262.

An isoform of this enzyme, referred to herein as deoxyribonuclease IIβ(DNase IIβ) has now been identified.

This full length gene for this isoform was first identified in mice bysequence comparison to expressed sequence tags entered in Genbankdatabase which were similar, but not identical to DNase IIα.Oligonucleotide primers were synthesized to obtain the complete DNaseIIβ mouse gene. The mouse DNase IIβ cDNA sequence is depicted as SEQ IDNO:1. The protein sequence of mouse DNase IIβ is depicted in SEQ IDNO:2.

Information from the mouse sequence was used to isolate a human homologof this gene. The human DNase IIβ cDNA sequence is depicted as SEQ IDNO:3. The protein sequence of human DNase IIβ encoded by the cDNA of SEQID NO:3 is depicted in SEQ ID NO:4.

Mouse and rat cDNAs of this homolog of DNase IIα have also beendisclosed recently by Shiokawa and Tanuma (Nucleic Acid Res. 199927(20):4083-4089 and Biochemical and Biophysical Research Communications1999 285:395-399).

It has been found that the DNase IIβ protein, like the DNase IIαprotein, retains a critical histidine in the predicted active site thusindicating that these proteins have similar activities. However, thereis sufficient difference in the region surrounding this histidine tosuggest that their activities, and in particular their potential as atherapeutic for cystic fibrosis, may be slightly different.Specifically, the predicted active site of human DNase IIα isFNSTEDHSKWCV (SEQ ID NO:5) while the equivalent sequence in the humanDNase IIβ isoform is FSSYQDHAKWCI (SEQ ID NO:6).

Further, it has now been found that DNase IIβ is expressed at highlevels in human salivary glands and is secreted into the saliva.

Using fluorescence in situ hybridization (FISH), it has now beendetermined that the human DNase IIβ is located at chromosome 1p22.Chromosome 1p22 is frequently a lost or rearranged region in numeroustypes of cancer including breast, lymphoma, liver and mesothelioma.While several genes in this region have been investigated, no clearcandidate for the tumor suppressor at this locus has been identified.DNase IIα is lethal when reintroduced into cells. Based on sequencesimilarity, it is expected that its isomer DNase IIβ will have similaractivity. Since this cell killing activity is consistent with thefunction of tumor suppressor genes, it is believed that DNase IIβ couldrepresent the tumor suppressor that is lost in these types of tumors.Accordingly, the mouse and human DNase IIβ gene sequence and protein ofthe present invention are believed to be useful in the development ofassays, screening approaches and targeted therapies for cancer.

For example, polymerase chain reaction (PCR) techniques can be used todetermine whether the gene is missing or mutated in cancer cells. Suchcells are expected to be more susceptible to the introduction of foreigngenes through means such as gene therapy.

Identification of agents which increase DNase IIβ expression areexpected to be useful in suppressing tumor formation and/or inducingapoptosis in cells. Inducing apoptosis is not only useful in treatmentof cancer, but also in the treatment of various autoimmune disorderssuch as multiple sclerosis in which immune cells that recognize thenormal patient tissue have failed to die as should normally happen.

The mouse and human DNase IIβ gene sequence and protein of the presentinvention are also useful in the development of agents which decreaseexpression of endogenous DNase IIβ in cells. For example, antisenseagents targeted to a portion of the cDNA sequence of the presentinvention or the corresponding mRNA can be developed. These antisenseagents can then be used to decrease or inhibit the expression of DNaseIIβ thereby protect cells from premature death. These antisense agentsmay therefore be useful in treating diseases resulting from too muchcell death such as neurodegeneration and AIDS.

Accordingly, cDNAs of the present invention are useful in identifyingagents which modulate, i.e., increase or decrease, apoptosis in cells.In this method, cells from a single culture are divided in two groups.The first group, referred to as the treated cells, are placed in contactwith a test agent in a vehicle. The second group, referred to asuntreated cells, are placed in contact with vehicle only. Treated anduntreated cells are then transfected with the cDNA of the presentinvention and apoptosis in the treated and untreated cells is monitoredto determine whether treating cells with the test agent modulatesapoptosis in the cells.

In addition, the DNase IIβ proteins of the present invention orfragments thereof are useful as antigens to produce antibodies thereto.By “antibody” it is meant to include, but is not limited to, bothpolyclonal and monoclonal antibodies as well as chimeric, single chain,and humanized antibodies along with Fab fragments, or the product of aFab expression library. Various techniques for producing such antibodiesare well known in the art.

Polyclonal antibodies generated against DNase IIβ can be obtained bydirect injection of the isolated, purified proteins of the presentinvention or fragments thereof into an animal, preferably a nonhuman.Such antibodies can then be used to isolate the enzyme from tissuesexpressing that enzyme.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used. Suchtechniques are used routinely by those skilled in the art. Some examplesinclude, but are not limited to, the hybridoma technique, the triomatechnique, the human B-cell hybridoma technique and the EBV-hybridomatechnique.

These antibodies are useful in studying the expression of DNase IIβ in avariety of cells. DNase IIβ levels can be determined in selected cellsby contacting selected cells with the antibody against DNase IIβ anddetecting binding of antibody to deoxyribonuclease IIβ enzyme in theselected cells. For example, in one embodiment, an antibody of thepresent invention is used to detect the intact protein in normal humancells compared to tumor cells to determine whether the tumor cells failto express the endonuclease.

DNase IIα digests DNA. Thus, given the similarity between DNase IIα andthe IIβ isoform of the present invention, it is believed that DNase IIβwill also digest DNA. Patients suffering from cystic fibrosis haveviscous sputum in their lungs; accumulation of this viscous sputum canlead to suffocation. Much of this viscosity comes from the release ofDNA from cells dying in the lungs. DNase I is currently used in patientswith cystic fibrosis as an inhaler to digest DNA in the mucous plugs ofthe lungs of these patients. However, this enzyme is inhibited by actin,also present in sputum. Thus, the efficacy of this treatment is limited.Previously, DNase II enzymes would not have been considered a practicalalternative because enzymatic activity was only observed at a pH belowthat of the lungs. However, the low pH activity of DNase IIα isassociated with a small DNase II fragment rather than the full lengthprotein. The full length DNA IIα and DNA IIβ identified herein may haveother catalytic activities such as an ability to digest DNA at higherpH. Accordingly, it is believed that administration of a concentrationof a DNase II enzyme which causes digestion of DNA in sputum will beeffective in alleviating suffering of patients with cystic fibrosis bydecreasing the viscosity of the sputum in the lungs.

The following nonlimiting examples are provided to further illustratethe present invention.

EXAMPLES Example 1 Identification of Expressed Sequence Tags

The cDNA sequence of DNase IIα was submitted to the Genbank database ona regular basis for analysis against the rapidly accumulating datadeposited therein to identify other cDNA and protein sequences withsimilarity to DNase IIα. An expressed sequence tag (EST) from mouse cDNAwas identified that has high similarity to DNase IIα. These ESTsequences are random pieces of cDNA that have been partially sequencebut have no known function. The identified mouse EST was purchased andcompletely sequenced. This sequencing revealed a complete cDNA sequencewith considerable homology to DNase IIα, but with sufficient differencesthat it obviously represented a different gene.

Additional EST sequences from human tissues were found that hadsimilarity to this mouse EST. However, upon sequencing they containedincomplete sequences. Specifically, EST #AI420898, whose sequence wasdeposited into Genbank on Mar. 28, 1999 was found to contain 932 bp ofthe gene referred to herein now as DNase IIβ. This sequence was clonedinto pT7T3D-Pac vector from Pharmacia.

Example 2 Nucleic Acid Sequencing

Plasmid DNA obtained in Example 1 was sequenced using the Big-DyeDeoxyTerminator Cycle Sequencing Kit from Applied Biosystems, followed byanalysis on an Applied Biosystems 370 DNA automated sequencer.

Example 3 Genomic Localization

Human genomic DNA was used as a substrate for PCR using oligonucleotideprimers predicted from the homology with DNase IIα to span intron 5 ofDNase IIβ. A 2,000 base pair fragment was isolated and cloned into thePCR-script vector. This genomic fragment was biotinylated and used as aprobe in fluorescent in situ hybridization to whole chromosomes. Theprobe hybridized to chromosome 1p22.

1. A cDNA encoding the deoxyribonuclease IIβ enzyme comprising SEQ IDNO: 1 or
 3. 2. A vector comprising a cDNA of claim 1.